Radioligands for the TRP-M8 receptor and methods therewith

ABSTRACT

One embodiment of the invention is a composition that comprises a radioactive [ 18 F], [ 76 Br]-, [ 77 Br]-, [ 211 At]-, [ 123 I], [ 125 I], or [ 131 I]-N-radioistope-labeled-aryl-alkyl-alkylcarboxamide molecule. The composition binds to the transient receptor potential-M8 (TRP-M8) receptor of cells. The TRP-M8 receptor is selectively expressed in sensory neurons and in malignant tissues such as prostate cancer cells. The [ 18 F], [ 76 Br]-, [ 77 Br]-, [ 211 At]-, [ 123 I], [ 125 I], or [ 131 I]-N-radioistope-labeled-aryl-alkyl-alkylcarboxamide ligand may be used for radioreceptor binding studies, for diagnostic studies, and for radiotherapy of cancerous tissues. Affinity of the N-radioistope-labeled-aryl-alkyl-alkylcarboxamide ligand for the TRP-M8 receptor confers selectivity and specificity in delivering lethal radiation to the diseased cells.

This application is a continuation-in-part of Ser. No. 10/687,188, filedOct. 15, 2003, Inventor Wei, entitled “Radioligands for the TRP-M8Receptor and Methods Therewith”, incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to chemicals that bind to receptors inthe TRP (transient receptor potential) ion channel family, moreparticularly to the subgroup of long TRP (or TRPM) channels, and mostparticularly to those that specifically bind to the TRP channel calledTRP-M8 (trp-p8, CMR₁); TRP-M8 receptors are present in sensory nervesand activation of these receptors is associated with cool and coldsensations. These receptors are also at elevated levels in the tissuesof certain cancers, such as prostate and breast cancer. This inventionmore particularly relates to TRP binding compositions containingradioisotopes such as radioactive fluorine and iodine ¹⁸F, ¹²³I, ¹²⁵I,or ¹³¹I, within the molecular structure, said compositions being useful,for example, in radioreceptor, diagnostic imaging, and radiotherapeuticapplications.

2. Description of Related Art

About two decades ago a group of scientists discovered novel compoundsthat have a physiological cooling action on the skin. These weredescribed in U.S. Pat. No. 4,193,936 (Watson et al., Mar. 18, 1980),U.S. Pat. No. 4,248,859 (Rowsell et al, Feb. 3, 1981) and U.S. Pat No.4,318,900 (Rowsell, Mar. 9, 1982). Much more recently a newphysiological receptor was discovered. This 1104-amino acid protein,deciphered from the cDNA sequence, was named trp-p8 because of itsstructural homology to receptors of the transient receptor potential(TRP) family. The mRNA for the synthesis of this specific protein wasalso detected in samples of malignant prostate, mammary gland cells,melanoma, and colorectal cancer cells. The functional role, if any, ofTRP-M8 receptors on malignant cells is not known.

The TRP-M8 sequence of the gene/protein was published in Cancer Research(vol. 61, pg. 3760-3769, May 1, 2001. L. Tsavaler, M. H. Shapero, S.Morkowski, and R. Laus: “Trp-p8, a novel prostate-specific gene, isup-regulated in prostate cancer and other malignancies and shares highhomology with transient receptor potential calcium channel proteins”).Soon afterwards it was discovered that this receptor was present insensory neurons and transduced the sensations of cold temperatures(McKemy et al. “Identification of a cold receptor reveals a general rolefor TRP channels in thermosensation”. Nature 416: 52-58, March 2002).Chemicals that elicit sensations of cold, such as menthol and icilin,bind to and activate the cold receptor, as measured by binding constantsand by calcium influxes into the cells.

A nomenclature panel composed of experts in the field has recommendedthe TRP-M8 designation for the cold/prostate receptor because of itsstructural homology to other protein receptors in this family. However,some still call this receptor trp-p8 or CMR₁ (cold-menthol receptor).The tags for the TRP-M8 sequences in the NicePro TrEMBL Database areQ8R405 (mouse TRP-M8), Q8R444 (rat TRP-M8 or CMR₁) and Q8TAC3 (humanTRP-M8, or trp-p8). The corresponding identity tags in the GenBank areAF4811480 and AY095352 (mouse), AY072788 (rat) and AY090109 (humans).

Various radioactive fluorine and iodine compounds are used in clinicaloncology. For example, ¹⁸F and ¹²³I are used in positron emissiontomography (PET) and single-photon emission computed tomography (SPECT),respectively, for the imaging, diagnosis and staging of neoplasticdisease. ¹²⁵I and ¹³¹I are used for the treatment of cancer, especiallythyroid cancer. Radioiodine compounds in thyroid therapy are remarkablyeffective because iodine is incorporated specifically into the thyroidhormones (thyroxin and tri-iodothyronine). Hence, the malignant cellsare selectively and specifically targeted, with minimal damage to normalcells and adverse side effects.

Prostate cancer is the most common cancer among men in the UnitedStates. There is no universally agreed-upon strategic plan for itsdiagnosis and management. Brachytherapy, a treatment well known in theart, involves the implantation of radioactive seeds directly into theprostate gland. The radioactive seeds used in brachytherapy may includeiodine-125, iodine-131, palladium, radium, iridium, or cesium. Anothercommon cancer is bladder malignancy, which will be diagnosed in anestimated 44,640 men and 15,600 women in the United State in 2004, withabout 13,000 deaths from this disease.

The pharmacological strategy, to bring radio-labeled compounds tospecific targets in malignant cells, to improve diagnosis, or to treatcertain cancers, is called targeted radiodiagnostics and targetedradiotherapy. New radiofluorinated and radioiodinated compounds usefulfor these applications are being sought.

BRIEF SUMMARY OF THE INVENTION

The present discovery provides carboxamide ligands that are usefullylabeled with various radionuclides, and which have a high affinity toTRP-M8 receptors in cells and tissues. Formula 1 illustrates carboxamideligands of this discovery.R—(C═O)—N(H or CH₃)—R′—Y  Formula 1

-   -   where (a) R is a branched hydrophobic carbon unit with 5 to        about 14 carbon atoms, and is preferably a cycloalkane radical        with one to three C₁ to C₅ normal or branched alkyl        substituents,        -   (b) R′ is an optional carbon bridge having C₁-C₃ carbons            which may include a hydroxy group, and        -   (c) Y is an aromatic radical containing at least one            substituent selected from R₁ and R₂, and at least one            substituent X, wherein            -   R₁, is selected from the group hydrogen, hydroxyl, C₁-C₅                alkyl, C₁-C₃ alkoxy, C₁-C₃ carboxyalkyl, C₁-C₄                carbonylalkylester, C₁-C₃ oxycarbonylalkyl, C₁-C₃                hydroxyalkyl,            -   R₂ is selected from the group —SO₂NH-pyrimidine,                —SO₃—(H, Me or Et), or —CH₂—SO₃—(H, Me or Et), acetyl,                C₁-C₃ hydroxyalkyl, trifluoromethyl, nitro, cyano, halo,                and            -   X is selected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—,                [¹³¹I]—[⁷⁶Br]—[⁷⁷Br]— and [²¹¹At]—.

In one aspect of the present invention, N-radiohaloaryl-alkylcarboxamideradioligand embodiments with specific affinity for the TRP-M8 receptorare provided (where there is no carbon bridge, R′, of Formula 1). Theseradioactive ligands are useful to study receptor binding (and toidentify new drugs that activate the TRP-M8 receptor), to conductradioimaging and radiodiagnostics, and should be useful as radioligandsfor therapy. The radionuclides preferred are ¹⁸F, ¹²³I 125I, or ¹³¹I.The inventive [¹²⁵I]-compounds are useful for laboratory tests, calledradioreceptor assays. The inventive [¹⁸F], [¹²³I], [¹³¹I]-compounds areuseful for imaging of the tumor cells in vivo bearing this receptormarker, and the [¹²⁵I] or [¹³¹I]-compounds are further believedpotentially useful for targeted radiotherapy.

Among particularly preferred compositions of this first embodiment arethose including [^(123, 125), or¹³¹I]—N-(4′-iodo-2′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamideillustrated below as Structure 1.

Also among particularly preferred compositions of the first embodimentare those that include[¹⁸F]—N-(4′-fluoro-2′-hydroxyphenyl)-2-isopropyl-2,3-dimethylbutyramideillustrated as Structure 2.

In another aspect of this invention, N-radioisotope-labelledaryl-alkyl-alkylcarboxamide radioligands with specific affinity for theTRP-M8 receptor are provided (where the carbon bridge of R′ has one, twoor three carbons). Among particularly preferred compositions of thesecond embodiment are those that include the compound illustrated byStructure 3.

In this Structure 3 compound, R′ is hydroxyethyl, R₁ is acetyl or—SO₂NH-pyrimidine, and R₂ is hydroxyl or hydroxymethyl, and X isselected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—, [¹³¹I]— [⁷⁶Br]—[⁷⁷Br]— and [²¹¹At]—

In yet another aspect of the present invention, methods are provided inwhich a Formula 1 compound having a determinable binding for the TRP-M8receptor and having a specific activity of about 20 Ci/mmol or greateris exposed to or contacted with a plurality of TRP-M8 receptors underconditions sufficient to permit specific binding therebetween. Thesemethods include radioreceptor assays, diagnostic imaging andradiotherapy, particularly for the diagnosis, monitoring and potentialtherapy of prostate cancer.

Other advantages and aspects of the present invention will be understoodby reading the following detailed description and the accompanyingclaims.

DETAILED DESCRIPTION OF THE INVENTION

With reference generally to Formula 1 below, radioligands of theinvention have a) a hydrogen bonding site, as exemplified by the CO andNH groups of a carboxamide, b) a hydrophobic group, as exemplified bycycloalkyl or branched aliphatic groups, and c) an aryl group that canbe halogenated with radioisotopes. The hydrogen bond/hydrophobic carbonunits optimize docking into the TRP-M8 binding site, and the aryl ringpermits delivery of the isotope.

The selected isotopes, preferably from the group ¹⁸F, ¹²³I, ¹²⁵I, and¹³¹I, serve to either mark the location or quantity of the TRP-M8receptor or to deliver radiation to the TRP-M8 bearing cell.

Embodiments of the present invention can function as ligands for theTRP-M8 receptor and preferably have high affinity to TRP-M8 sites incells and tissues and a specific activity of about 20 Ci/mmol orgreater.

Compositions including the inventive radioligands of the invention havethe following applications:

-   -   use as ligands for TRP-M8 radioreceptor assays in the        laboratory;    -   use as ligands for diagnosis and imaging of TRP-M8 receptors in        prostate tissues and cells; and    -   use as radiotherapeutics (alone or co-administered with local        anesthetic amidase inhibitors as potentiators) for prostate        disorders such as cancer or benign hyperplasia.

Formula 1 illustrates carboxamide ligands of this discovery.R—(C═O)—N(H or CH₃)—R′—Y  Formula 1

-   -   where (a) R is a hydrophobic alkyl radical, more particularly a        branched hydrophobic carbon unit with 5 to about 14 carbon        atoms, and more preferably is a cycloalkane radical with one to        three C₁ to C₅ normal or branched alkyl substituents,        -   (b) R′ is an optional carbon bridge having C₁-C₃ carbons and            which may include a hydroxy group, and        -   (c) Y is an aromatic radical containing at least one            substituent selected from R₁ and R₂, and at least one            substituent X, wherein            -   R₁ is selected from the group hydrogen, hydroxyl, C₁-C₅                alkyl, C₁-C₃ alkoxy, C₁-C₃ carboxyalkyl, C₁-C₄                carbonylalkylester, C₁-C₃ oxycarbonylalkyl, C₁-C₃                hydroxyalkyl,            -   R₂ is selected from the group —SO₂NH-pyrimidine,                —SO₃—(H, Me or Et), or —CH₂—SO₃—(H, Me or Et), acetyl,                C₁-C₃ hydroxyalkyl, trifluoromethyl, nitro, cyano, halo,                and            -   X is selected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—,                [¹³¹I] [⁷⁶Br]— [⁷⁷Br]— and [²¹¹At]—.

Where R′ has no carbons (that is, R′ is not present), then preferredembodiment compositions are those comprising a radioactive compoundhaving the structure [¹⁸F], [¹²³I], [¹²⁵I], or[¹³¹I]-N-radiohaloaryl-alkylcarboxamide, for example:[^(123, 125 or 131)I]—N-(4′-iodo-2′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide,and[¹⁸F]—N-(4′-fluoro-2′-hydroxyphenyl)-2-isopropyl-2,3-dimethylbutyramide,illustrated below as Structures 1 and 2 respectively.

Where R′ is present, then among particularly preferred compositions ofthe second embodiment are those that include the compound illustrated byStructure 3.

In Structure 3, R′ is hydroxyethyl, R₁ is acetyl or —SO₂ NH-pyrimidine,and R₂ is hydroxyl or hydroxymethyl, and X is selected from the group[¹⁸F]—, [¹²³I]—, [¹²⁵I]—, [¹³¹I]—, [⁷⁶Br]—, [⁷⁷Br]—, and [²¹¹At]—.

The term “alkyl” used throughout this description in the context of theR group of Formula 1 is as a generic term to include both acyclic alkylgroups and cycloalkanes. That is, the hydrophobic group is provided by abranched hydrophobic carbon unit, which can be supplied by cycloalkyl orbranched aliphatic groups. However, cycloalkyl groups, particularlythose where R is a cycloalkane derivative of cyclopentanes,cyclohexanes, cycloheptanes, and cyclooctanes, are preferred.

Radioactive compounds of the present invention preferably have aspecific activity of at least about 20 Ci/mmol, more preferably have aspecific activity of at least about 250 Ci/mmol. Radioactive compoundsof the invention can function as ligands for the TRP-M8 receptor, andpreferably have a Kd for the receptor of about 1×10⁻¹² to 1×10⁻⁵ molar.

An aspect of the present invention is that the radionuclide (preferably¹⁸F, ¹²³I, ¹²⁵I or ¹³¹I) is incorporated (i.e. covalently bound) withinthe molecular structure of the ligand for the receptor. One advantage ofthis is that the radiation emitted can readily be detected withradioactivity counters or imaging systems and is directly correlated tohigh affinity binding to TRP-M8 receptors. Such specific directradioactive label incorporation into the binding molecule is uncommonand provides excellent results in radioreceptor applications ascontemplated in the present invention.

By contrast, a laboratory procedure, for example, the labeling of abinding protein such as a monoclonal antibody by ¹²⁵I, carries the riskthat the protein will be denatured by the iodine and degraded byenzymes, thereby reducing or destroying its high affinity binding to thereceptor target. Moreover, the points of attachment of iodine to thebinding molecule are non-specific (see Griffiths et al,. Radioactiveiodine labeled proteins for targeted radiotherapy, U.S. Pat. No.5,976,492, Nov. 2, 1999, herein incorporated by reference).

Incorporating ¹⁸F, ¹²³I, ¹²⁵I or ¹³¹I by covalent binding into moleculesof the present invention avoids the drawbacks referred to above withradioactive iodine with respect to denaturation, degradation, andpotential loss of activity, since incorporation of the radioisotope intothe molecule does not significantly change the physical-chemicalproperties of the molecule. The chemical features of the molecule thatdetermine specificity of binding affinity are retained, with the addedproperty of radiation. Compounds of the invention are sometimes hereincollectively termed “N-radiohaloaryl-carboxamides”.

Criteria for Bloactivity on TRP-M8

In Vivo Assays for TRP-M8 Activation. The endogenous TRP-M8 receptor isa physiological receptor designed to detect temperature changes in itsenvironment and to transmit this signal to the central nervous system sothat appropriate regulatory responses can be initiated (e.g.vasoconstriction to reduce heat loss, putting on warmer clothing,avoiding the cold environment). This receptor also responds to drugligands (e.g., menthol, icilin, certain N-alkylcarboxyl esters andN-alkylcarboxamides) which activate its message transmission system andelicit sensations of cold.

The TRP-M8 receptor on cold sensory nerve endings and on malignantcells, for example in the prostate, are biochemically identicalproteins. Thus, the potency of a molecule to elicit cold sensations, forexample on the tongue or skin, was used as surrogate index of thebinding affinity of the molecule for the TRP-M8 receptor. A number ofpotent “N-radiohaloaryl-carboxamides” were synthesized and tested foruse as precursors of the inventive compounds and the results are shownin Table 1. TABLE 1 Cold Sensation Threshold on CHEMICAL Tongue*, μgN-(3′-hydroxy-4′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide0.1 N-(4′-methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 0.1N-(2′,4′-dimethylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 0.1N-(4′-methoxy-2′-methylphenyl)-1-isopropylcycloheptanecarboxamide 0.2N-(4′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 0.3N-(4′-nitrophenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 0.3N-(2′-hydroxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 0.5N-(4′-fluorophenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 0.5N-(4′-methoxyphenyl)-2-isopropyl-2,3-dimethylbutyramide 0.5N-(3′-hydroxy-4′-methylphenyl)-1-isopropylcycloheptanecarboxamide 1N-(4′-hydroxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 1N-(2′,4′-dimethylphenyl)-2-isopropyl-2,3-dimethylbutyramide 1N-(4′-acetylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 2N-(4′-methoxyphenyl)-2-isopropyl-2,4-dimethylpentanamide 2N-(4′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 3N-(3′,4′-dimethylphenyl)-2-isopropyl-2,3-dimethylbutyramide 3N-(3′,4′-dimethoxyphenyl)-1-isopropylcycloheptanecarboxamide 5N-(4′-ethoxycarbonylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 5N-(4′-methoxyphenyl)-1-ethyl-2-methylcycloheptanecarboxamide 6N-(4′-chlorophenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 8N-(2′,4′-dimethylphenyl)-1-isopropylcycloheptanecarboxamide 15N-(3′,4′-dimethylphenyl)-1-isopropylcycloheptanecarboxamide 15N-phenyl-2-isopropyl-5-methylcyclohexanecarboxamide 20N-phenylmethyl-2-isopropyl-5-methylcyclohexanecarboxamide 20*Filter paper (1 × 1 cm) was impregnated with a known amount of compoundand placed on the tongue of the test subject. After 30 sec, the subjectwas required only to report presence or absence of a cooling effect.Individual sensitivity varied over a considerable range; for example,for 23 subjects, chosen at random, the threshold for a standard such asmenthol ranged from 0.02 to 10 μg. Ethoxycarbonyl is COOCH₂H₅.Cooling Actions on Skin. CPS-195[2-Isopropyl-5-methyl-cyclohexanecaboxylic acid[2-hydroxy-2-(3-hydroxy-phenyl)-ethyl]-methyl-amide], CPS-140[2-Isopropyl-5-methyl-cyclohexanecaboxylic acid [4-acetylphenyl]-amide]and CPS-125 [2-Isopropyl-5-methyl-cyclohexanecarboxylic acid[4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide], dissolved 0.5 to 2% wt/volin Aquaphor® ointment and then applied to the surface of the philtrum ofvolunteer human subjects produced cooling sensations lasting from 1.5 to2.5 hr. These results show that compounds of this application canactivate the TRP-M8 receptors.In Vitro Assays for TRP-M8 Activation. The methods for the TRP-M8receptor studies, using methods of calcium ion imaging or intracellularvoltage changes, are described in Behrendt et al. [Characterization ofthe mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1using a fluorometric imaging plate reader (FLIPR) assay. Brit. JPharmacol. 2004 Feb; 141(4):737-45], and by A. K. Vogt-Eisele, D. Bura,H. Hatt, and E. T. Wei. [N-Alkylcarboxamide Cooling Agents: Activitieson Skin and Cells with TRPM8 and TRPA1 Receptors. Acta Dermato-Venereol.85: 468, 2005.] The data here were collected for the applicant by Dr.Afrodite Lourbakos of Unilever Research and Development, theNetherlands, using similar transfection methods and a FLIPR assaysystem. For the patch-clamp studies, using measurements of intracellularvoltage changes, some of the data reported here were collected by Dr.Matthias Bödding of the University of the Saarland, and by Dr. AngelaVogt-Eisele of the Ruhr University at Bochum, Germany.FLIPR assay Human embryonic kidney (HEK) cells were permanentlytrausfected with plasmids containging the cDNA coding for the gene forthe human TRP-M8 receptor. These cells were then incubated with acalcium fluorescence indicator (Fura-2) and incubated at either 29 or37° C. These cells were then distributed into a 96-wellfluorescence-plate image reader with automated drug dilution andcomputerized software for dose-response analysis. Cacium ion influx intocells after stimulation with compounds was quantified in fluorescenceunits. Compounds were dissolved in DMSO by ultrasonication to a 0.1 Msolution. 5 μl of this stock was added to 5 mg of cyclodextrin and 5 mlof 140 Na-Tyrode, to achieve various final test concentrations. Icilinand menthol, standard TRP-M8 agonists, were used as positive controlsand gave median effective concentration activities of 0.8 and 25 μM(EC50) activities. The EC50 of various test substances are shown inTable 2. Further analysis of the dose-response relationship showed theΔFmax (the maximum fluorescence increase induced by a compound at themaximum concentration tested) for various compounds to be at the maximumof 14,000 units which was similar to that seen with icilin and menthol,confirming full activation of the TRP-M8 receptor.

Patch-Clamp Electrophysiological Recordings. HEK cells were prepared asabove. Membrane currents were recorded in the whole-cell configurationusing an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany) asdescribed previously (Bödding et al. 2002). Patch pipettes pulled fromborosilicate glass (Kimax®) had resistances between 2 and 3 MΩ whenfilled with the standard internal solution (in mM): 145 Cs-glutamate, 10HEPES, 8 NaCl, 1 MgCl₂, 2 Mg-ATP, 0.1 mM EGTA adjusted to pH 7.2 withCsOH. The EGTA concentration was 10 mM for the experiments shown in FIG.6. Extracellular solution contained (in mM): 145 NaCl, 2 CaCl₂, 2.8 KCl,2 MgCl₂, 11 glucose, 10 HEPES, adjusted to pH 7.2 with NaOH. Drugs wereapplied in the bath solution by a custom-made local perfusion system.The series resistance was compensated for 80 % and ranged between 5 and10 MΩ. Currents were filtered using an 8-pole Bessel filter at 2.9 kHzand digitised at 100 μs. Voltage ramps (−110 mV to 90 mV in 50 ms) wereapplied at 0.5 Hz from a holding potential of −10 mV using PULSEsoftware (HEKA Electronics). Several parameters such as capacitance,series resistance and holding current were monitored simultaneously at aslower rate (2 Hz) using the X-Chart display (HEKA Electronics). Aliquid junction potential of 10 mV was applied to all voltages. Allexperiments were carried out at room temperature (20-23° C.). Internalsolutions were kept on ice to minimize hydrolysis of ATP. TABLE 2 EC50μM EC50 μM Code Names Structures FLIPR patch-clamp R-phenyl-X CPS-1284-OEt— 0.5 NA CPS-112 (WS-12) 4-OMe— 0.6 0.2 CPS-113 3-F, 4-OMe— 1.3 1.2CPS-124 4-fluoro- 1.3 1.2 CPS-129 2-iodo, 4-methoxy NA 0.3 CPS-1234-bromo- 6 NA CPS-120 4-iodo- 10 NA CPS-125 4-sulfadiazinyl- 6 3.0CPS-131 4-sulfadimethoxinyl- 60 NA CPS-141 3-OMe— 6 NA CPS-127 4-OCF₃—NR NA CPS-138 3-CF₃, 4-NO₂— NR NA CPS-132 4-sulfisoxazolyl- NR NA OtherStructures CPS-116 3-OMe, 4-OH-benzyl-R 8 NA CPS-195 3-OH,2-hydroxybenzyl- NA 0.1 N-methylated Comparison Cmpd menthol 25 10.4Comparison Cmpd icilin 0.8 1.4NR = no responses,NA = not availableStructure-Activity Relationships for Active Compounds. Precursorcompounds having the desired affinity for the TRP-M8 receptor (such aslisted in Tables 1 and 2) are radiohalogenated according to standardprocedures so as to form inventive comounds for uses in the presentinvention. The preferred [¹⁸F], [¹²³I], [¹²⁵I], or[¹³¹I]—N-halo-aryl-alkyl-alkylcarboxamides of the invention asillustrated by Formula 1. In addition to cycloalkanes, such as where theR of Formula 1 is (1R,2S,5R)-2-Isopropyl-5-methyl-cyclohexyl, R may alsobe a branched chain N-radioistope-labeled-aryl-alkyl-alkylcarboxamide.

Examples of such branched chains for R are propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl and pentyl, isopentyl, neo-pentyl etc.For branched aliphatics, the carboxamide is attached, for example, tothe “3” position of 2,3,4-trimethyl-pentane and 2,4-dimethyl-hexane, andto the “4” position of 3,5-dimethyl-heptane.

The structural features of TRP-M8 binding for N-fluoro- oriodo-aryl-alkyl-alkylcarboxamides side principally in the hydrogenbonding —C(═O)—NH— moiety and the branched chain hydrophobic carbonunit. The N-substituent can be quite varied; for example, N-ethyl orN-methyl p-menthane-3-carboxamides have oral cooling thresholds as lowas 0.2 to 1.1 μg, respectively. In receptor terminology, theN-substituent portion of the molecule is “promiscuous” and manyalternatives are permissible to fit the TRP-M8 receptor pocket. Thus,the “Y” of Formulas 1 and 2 can be a substituted aromatic radical,selected from the group phenyl, benzyl, 1-naphthyl, 2-naphthyl,1-anthracenyl, 2-anthracenyl, 9-anthracenyl, as well as otherpolyaromatic aromatic rings such as indene, azulene, heptalene,indacene, acenapthlene, fluroene, phenanthrene, and further as well asheterocyclic aromatic rings such as pyridine, dihydropyridine,pyridazine, pyrimidine, pyrazine, indole, purine, indolizine, quinoline,isoquinoline, quinazoline, carbazole, phenazine, phenothiazine, andphenathridine. A polyaromatic ring will also permit multiplehalogenation which increases the specific activity of the TRP-M8 ligandand enhances measurement of binding, imaging, and delivery of radiation.

Radiohalogenation of aromatic rings to generate ligands for receptorbinding, for radioimaging and for radiotherapy is a chemical techniquethat is familiar to many practioners of the art. The preferred isotopes,¹⁸F, ¹²³I, ¹²⁵I, and ¹³¹I, are most commonly used but it should be notedthat alternative therapeutic radionuclides are also contemplated. Forexample, for targeted radiotherapy for small tumors other halogens suchas ⁷⁶Br and ⁷⁷Br, and low-energy electron emitters such as ^(58m)Co,^(103m)Rh, ¹¹⁹Sb, ¹⁶¹Ho, and ^(189m)Os are also feasible (Bernhardt etal. Low-energy electron emitters for targeted radiotherapy for smalltumours. Acta Oncologica 40: 602-608, 2001). Radionuclides, such as²¹²Bi, ²¹³Bi and ²¹¹At, a halogen, which decay by the emission ofalpha-particles, can also be incorporated into the N-aryl-alkyl moietyand are attractive for applications of targeted radiotherapy inaccordance with the present invention. The halogens, such as Br and At,may be attached using trialkyl tin reagents and the metal isotopes, suchas Sb and Os, may be attached to the ring using metal chelating agents.

Precursor compounds of highest activity and especially preferred for usein the present invention (after modification to incorporateradiohalides) are described in Table 1 and further illustrated by theexemplary preparation of Example 1.

The particular N-substituent portion of the molecule, namely the —R′—Yof Formula 1, would vary depend upon the particular laboratory,radiodiagnostic, or radiotherapeutic applications. For example, inlaboratory applications, the ideal isotope will be ¹²⁵I and selection ofthe ideal probe for the receptor will be based on potency of activationor binding affinity. In radiodiagnostic applications, for example, inPET scanning, the isotope will be ¹⁸F, but here the pharmacolineticbehavior of the prototype needs to be considered because it has to bedistributed in the vascular compartiment and access the target tissuessuch as prostate or breast cancer cells. In such molecules, insertion ofoxygenated functions, such as hydroxy ethyl will lower octanol/waterpartition coeffcients and facilitate access of the designed molecule toreach target.

If the target is designed for killing cancer cells in the bladderurothelium, the delivery would be via urine of a drug molecule that hasa) has high affinity binding to the TRP-M8 receptor, on the order of aKd of 10⁻⁹ M or less, b) contains lethal radiation in the form of analpha, beta or gamma emission from a radioisotope within its molecularstructure, and c) is water-soluble, because urine is an aqueous media.The drug may given by oral or by parenteral administration, e.g. byintravenous injection or the may also be delivered directly into thebladder lumen with a transurethral catheter or by intravesicalinjection.

For the urothelium, if delivered by oral or by parenteraladministration, the “letter bomb” drug preferably enters the bloodstreamand then is cleared and concentrated by the kidneys into the urine. Thecreation of such a drug requires ingenuity in design. Ideally, this drugwould be: a) relatively polar and water-soluble and thus filtered andconcentrated into the urine, b) of a molecular weight under 500 daltonsso that it readily passes through the glomeruli and tubules c) of a pKaof 4.5 to 7.5, so that it is significantly ionized at urinary pH, d)minimally bound to protein in the blood so that it can be filtered, ande) not be actively re-absorbed by the renal tubules. Overall, >90% ofthe drug should preferably be cleared within 24 hr, to minimizeirradiation of non-cancerous tissues. Finally, the drug must retain itsselective high affinity for the TRP-M8 on the urothelium.

In Formula 1, the left-hand moiety R—(C═O)—N (H or CH_(3v)) confersstructural features for TRP-M8 binding. The branched chain hydrophobiccarbon unit and the amide feature are essential for receptor fit, withmolecules having Kd in the range of 10⁻⁷ to 10⁻⁶ M. The N-substituentcan be quite varied but a 100 to 1000-fold increase in potency isachieved when a phenyl or aryl with an oxygen atom containing function(e.g. para-methoxy or sulfadiazine). With these substituents, the goalof potencies in the range of 10⁻⁸ to 10⁻¹⁰ M is achieved. TheN-substituent portion of the molecule is sterically “promiscuous” andmany alternatives are permissible to fit the TRP-M8 receptor pocket, sothat the addition of a radioisotope (X) in the Formulae, such as ¹³¹I,does not substantially change affinity for the TRP-M8 receptor. Thepresence of the —R′— group permits the addition of polar groups whichfacilitate the delivery or pharmacokinetic profile of the radioligand totarget. In addition, the presence of one or more chiral centers on the—R′— carbon(s) allows the use of selective enantiomers to achievegreater receptor selectivity and specificity. This stereoisomerismpermits better selection of the radioligand.

Preparation of Inventive Compounds

The preparation of N-substituted-aryl-alkyl-alkylcarboxamides isfamiliar to practitioners of the art of chemistry and, for example, isdescribed in U.S. Pat. No. 4,193,936, incorporated by reference.Starting with the corresponding alkanoyl chloride, a single stepreaction with the appropriate amine yields the desired product. Forexample, an alicyclic compound, p-menthane-3-carboxylic acid (synonym:2-isopropyl-5-methylcyclohexanecarboxylic acid) is reacted with thionylchloride in diethylether to yield the p-menth-3-oyl chloride which, whenstirred with the substituted-aryl-alkylamine at room temperature forabout 4 hr, generates the correspondingN-substituted-aryl-alkyl-p-menthane-3-carboxamide. The precipitatedproduct is readily collected by filtration and may be recrystallizedusing solvents such ethyl acetate or purified on silica gel columns. Thefinal products are solids stable at room temperature.

The -aryl-alkylamine may, for example, be 3-methyl4-iodo-phenylamine, or4-fluorophenylamine, or 4-iodo-1-naphthylamine and the correspondingproduct after reaction with p-menth-3-oyl chloride would beN-(3′-methyl-4′-iodo-phenyl)-2-isopropyl-5-methylcyclohexane-3-carboxamide,N-(4′-fluorophenyl)-2-isopropyl-5-methylcyclohexane-3-carboxamide, andN-(4′-iodo-1′-naphthyl)-2-isopropyl-5-methylcyclohexane-3-carboxamiderespectively.

Synthesis of non-radioactive n-substituted-aryl-alkyl-alkylcarboxamidesas precursors for the inventive compounds are depicted in the followingschematics I and 2. Schematic 1 shows the synthesis of the desiredcarboxamide. Synthesis of radioactiven-substituted-aryl-alkyl-alkylcarboxamides useful in practicing thepresent invention to incorporate a halogen is accomplished with reagentsthat effect the halogenation process rapidly (because of the shorthalf-life of the isotopes). A standard reagent is trimethyl tin that isbonded to the site of radiohalogenation. Schematic 2 illustrates thishalogenation process with trimethyl tin in forming an embodiment of theinvention in which an analog of Structure 1 is formed with ¹⁸ F. In thisanalog, R₁ is hydrogen, R₂ is para-methoxy, and X is radioactivefluorine. This analog is active at nanomolar (10⁻⁹) in promoting calciumentry into TRP-M8 transfected cells and into LNCaP (lymph node prostatecancer cells) constitutively expressing these receptors.

Radioactive Ligands for the TRP-M8 Receptor.

The naturally occurring isotope of iodine has an atomic mass of 126.9Daltons. The radioactive isotopes of iodine are ¹²³I, ¹²⁵I and ¹³¹I withhalf-lives of 13.2 hr, 60.1 days and 8.0 days, and average energy ofradioactive emission of 0.159 Mev, 0.02 Mev and 0.36 Mev, respectively.Preparation of [¹²³I]-compounds require special facilities because ofthe short half-life of this isotope. By contrast, ¹²⁵I or ¹³¹I areinexpensive, readily available at high specific activity of severalCi/matom and obtainable by express mail. Iodine, being a commonconstituent of the body, has no inherent toxicity in its radioactiveform, other than the emitted radiation.

The natural isotope of fluorine has an atomic mass of 19.0. The¹⁸F-isotope has a half-life of 1.8 hr and an average energy of emissionof 0.511 Mev and requires special facilities for preparation. Thefluorine compounds used in this invention have no inherent toxicity atthe doses employed, other than the emitted radiation.

Amersham Biosciences Corporation (800 Centennial Avenue, Piscataway,N.J. 08855-1327, USA) is a major supplier of reagents for the synthesisof radio-labeled chemicals. Starting materials for [¹²⁵I]- or[¹³¹I]-compounds can be obtained from Amersham at high specificactivities. The isotope half-lives and the average energy of emissiondictate the practical use of these labels:

-   -   [¹²⁵I]-labeled alkylcarboxamides compounds are useful for        radioreceptor assays in the laboratory;    -   [¹⁸F]-, [¹²³I]- or [¹³¹I]-labeled        N-halo-aryl-alkyl-alkylcarboxamides are useful for scanning or        imaging tissues bearing the TRP-M8 receptor; and    -   [¹²⁵I]- or [¹¹³I]-labeled N-halo-aryl-alkyl-alkylcarboxamides        may be useful for targeted radiotherapy, using fractionated        dosages to destroy the desired amount of tissues.        Use of [¹²⁵I]-labeled N-halo-aryl-alkyl-alkylcarboxamides for        Receptor Assays

A TRP-M8 receptor has two integral components, an extracellular ligandbinding domain that detects the ligand signal and an intracellulardomain that is involved in signal transmission. The ligand detectionmechanism is structurally specific and analogous to the lock and keymodel of classical pharmacology. The key is the drug ligand and the lockis the receptor. Signal-transducing receptors are present in smallnumbers, on the order of a few thousand receptors per cell.Nevertheless, the receptors are designed to regulate crucial cellularfunctions and therefore become specific targets for drug discovery anddevelopment. Although not precisely understood, the amino acid residueson the TRP-M8 protein that correspond to ligand binding domains haverecently been identified. For example, aspartic acid on residue 802 andglycine on residue 805 of rat TRP-M8 are critical for icilin-inducedactivation of TRP-M8. Similarly, tyrosine on residue 845, tyrosine onresidue 1005, and leucine on residue 1009, influence menthol-inducedactivation of mouse TRP-M8.

To measure drug occupancy of the receptor, pharmacologists use the term“Kd (dissociation constant)” to represent the affinity of the drug toits receptor. The Kd is based on the molar concentration of the drugoccupying 50% of the receptor population, so the lower the Kd, thehigher the “affinity” or stickiness of the ligand for its receptor. Adrug receptor agonist, that is, a drug that elicits a biologicalresponse, generally has Kd values in the sub-micromolar (10⁻⁶),nanomolar (10⁻⁹) to picomolar (10⁻¹²) concentration and represents a“high affinity” binding site. Similarly, a drug that binds with highaffinity to the receptor, but which does not activate the receptor, maybe a high affinity antagonist, preventing the actions of an agonist. Tomeasure Kd for different chemicals, it is necessary to have a primaryradioligand that is chemically pure and stable and known to elicit thedesired receptor response (for TRP-M8, it can be the sensation of cold,or cation influx into transfected cells that express the receptor). Thespecific activity of the radioactive ligand must be high enough todetect high affinity binding of the receptor in the tissue beingstudied. This usually means a specific radioactivity of 30 Ci/mmol orhigher.

For example, a synthetic [¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamideligand, such as [¹²⁵I]-N-(4′-iodophenylethyl)-p-menthane-3-carboxamide(synonym:[¹²⁵I]-N-(4′-iodophenylethyl)-2-isopropyl-5-methylcyclohexanecarboxamide)is an excellent receptor-assay radioligand. Such a radioligand can besynthesized, for example, at 30 Ci/mmole, which is considerably belowthe theoretical maximum of 2000 Ci/mmole for [¹²⁵I]-labeled compounds.This ligand can then be used for radioreceptor assays of TRP-M8agonists, as illustrated by Example 2. A TRP-M8 receptor assay based on[¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand has severalapplications, as described infra.

Utility of Radio-Labeled -Alkylcarboxamides on TRP-M8 Receptor

An agonist, in pharmacological terminology, is a chemical that activatesbiological events. The agonist, almost by definition, acts on a specificbiological receptor to initiate cellular events. The purpose of aradioreceptor assay is to have methods to identify and measure ligandswith low Kd value, and hence high affinity for the desired receptor.Thus, in practice, the first step is the characterization of a prototype[¹²⁵I]-agonist of the TRP-M8 receptor. Once, a prototype has beenidentified, additional assays of in vitro and in vivo agonist activityare conducted to demonstrate that the binding is functional. Thesebioassays may also be conducted with non-radioactive alkylcarboxamidesto measure the median effective concentrations (EC50). These methods arestandard tools in drug screening.

An antagonist, in pharmacological terminology, is a chemical that bindswith high affinity to a receptor, occupies it, and prevents the actionsof an agonist; but the antagonist itself does not activate biologicalevents. A prototype [¹²⁵]-agonist of the TRP-M8 receptor can be utilizedin screening for a TRP-M8 receptor antagonist. Unknowns can be testedfor their ability to displace the [¹²⁵I]-agonist from its binding site.A high affinity antagonist would be a chemical that displaces the[¹²⁵I]-agonist at sub-micromolar concentrations, but by itself does notproduce cold sensations or activate TRP-M8 ion channels.

Carboxamide Radioligands for Laboratory and Diagnostic Applications.

The TRP-M8 receptor is exceptional in that its mRNA transcript is foundin abundance in biopsy samples of human malignant tissues such as breastcancer, colorectal cancer, melanoma and especially prostate cancer, butnot in normal tissues with the exception of prostate epithelial cells(Tsavaler et al., supra). The standard method used for detecting TRP-M8MRNA transcript in human tissues is to use in situ hybridizationtechniques with special riboprobes designed to detect the TRP-M8 cDNA.Serial sections of tissues are made, then stained, which enablehistopathologists to visually observe any TRP-M8 receptors in thestained tissue. Such methods, however, require advanced laboratoryskills and training. More recently, TRP-M8 antibodies have beendeveloped that allow for the detection of TRP-M8 receptor protein on thesurface of hyperplastic and malignant in prostate tissues. The use ofsaid antibodies (that is “TRP-M8 immunocytochemistry”) has confirmed thepresence of TRP-M8 on the surface of human prostate cancer cells.

A [¹²⁵]-radioreceptor assay in accordance with the present invention,designed to measure the amount and the presence of the TRP-M8 receptorprotein in biopsy samples, is potentially a less costly and a moreconvenient and a more direct alternative than the aforedescribedtechniques of in situ hybridization and TRP-M8 immunocytochemistry.

The radioreceptor assay technique has diagnostic applications forpatients having cancers that express the TRP-M8 receptor. For example, abiopsy sample of about 10 mg tissue may be homogenized and incubatedwith a [¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand for 30 min,centrifuged or filtered, dissolved in a solvent, and the beta-emissionscounted on a Geiger, scintillation, or other radioactive counter. Basedon the findings of Tsavaler et al, supra, one would expect a sharpincrease in the amount of TRP-M8 specific binding (Bmax) in malignanttissues, and the relative lower abundance of binding in normal tissues.Such measurements, with small amounts of tissue, because of thesensitivity of a radioreceptor method using a radioligand with highspecific activity, can be used, for example, to detect the presence ofdiseased tissues, to track disease progression, and to measuremetastases.

In the laboratory, ¹²⁵I is widely used in a technique calledautoradiography. Traditionally, ¹²⁵I, is used to label peptides orproteins in a non-specific location, such as the ring structure oftyrosine and on the ε-amino group of lysine. This technique permits thedetection and visualization of receptors or antigens that bind to thelabeled agonist or antibody. By the same principles, the[¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand may also be used inaccordance with this invention for autoradiographic studies of theTRP-M8 receptor and for discerning its role in hyperplastic andneoplastic processes.

For example, sections of prostate tissues may be incubated with theradioligand, rinsed, and then placed on X-ray film, and the precisesites of TRP-M8 localization mapped by autoradiography. The availabilityof the [¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide compositions of thepresent invention should facilitate the study of TRP-M8 expression inhyperplastic and malignant cells and aid in elucidating the role ofTRP-M8 in tumor initiation, transformation, invasiveness and metastaticactivity.

Radioimaging/Radiodiagnostic Uses of [¹⁸F], [¹²³I], [¹²⁴I] or[¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamides with High Affinity forTRP-M8.

Various radioactive fluorine and iodine compounds are used in clinicaloncology. For example, ¹⁸F and ¹²⁴I are used in positron emissiontomography (PET), and ¹²³I and ¹³¹I in single-photon emission computedtomography (SPECT), respectively, for the imaging, diagnosis and stagingof neoplastic disease. The emission of coincident or single high energyphotons permits computerized tomography imaging that yields usefulinformation about receptor marker binding, localization, and clearancerates. A useful isotope for PET imaging is ¹⁸F, an isotope with a 110min half life that generates coincident 511 KeV photons which ismeasured by PET at a resolution of 0.5 to 1.8 mm at markerconcentrations of 10⁻⁹ to 10⁻¹² M in tissues. A useful isotope for SPECTimaging is ¹²³I an gamma-emitter with a 13.3 hour half life. Eighty-fivepercent of the isotope's emissions are 159 KeV photons, which is readilymeasured by SPECT instrumentation currently in use.

The use of [¹⁸F]-deoxyglucose PET methods for monitoring the progress ofprostate cancer has limited success in part because such prostate cellshave limited metabolic activity. A [¹⁸F]-TRP-M8 ligand, using PETimaging, will have utility as a non-invasive method in staging thisdisease. ¹²⁴I, having a longer half-life of 4.11 days than ₁₈F can alsobe used, but it has limited availability. The high resolution of PET canalso allow the surgeon to detect metastases, to stage the disease, toassess hormonal sensitivity to androgens, and to gauge the feasibilityof tissue removal. Similarly, a [¹²³I] and [¹³¹I]-TRP-M8 ligand can beused for SPECT applications in prostate diseases.

Radiotherapeutic Use of [¹²⁵I] or[¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamides with High Affinity forTRP-M8.

The expression of TRP-M8 receptor in tissues of the prostate and bladderand its expression in hyperplastic and neoplastic conditions [Tsavaleret al. supra] makes this receptor a potential target for cancerradiotherapy. The TRP-M8 drug design strategy for this target must beselective and specific: selective in the sense that hyperplastic orcancer cells express this target more than normal cells, and specific inthe sense that the molecular target will have structural features thatbind the drug with high affinity. Standard pharmacological strategiesfor targeting receptors expressed in hyperplastic and neoplastic cellsare to:

-   a) make a monoclonal antibody against the target. The binding of the    monoclonal antibody to the receptor leads to cell death, for    example, by triggering apoptosis;-   b) make a small molecule agonist of the receptor to reactions that    cause cell death; and-   c) devise an epitope based on the receptor structure such that the    body will develop an antibody response and the immune system attack    against the receptor may reduce cancer growth.

I contemplate a fourth alternative. An isotopically-labeled TRP-M8receptor agonist or antagonist, for example, [¹²⁵I] or[¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamide, with high affinity bindingfor this receptor can be a “letter bomb” for killing cancer cellsbearing this receptor. Here, the binding affinity (that is, the addressto the receptor) is an innate part of the molecular framework and theradiation from ¹²⁵I or ¹³¹I is the lethal message. Unlike currentbrachytherapy technique, compounds and compositions of the presentinvention possess selectivity and specificity to deliver a sophisticatedlethal message to a specific target address. The targeted TRP-M8receptor may, for example, be in the prostate or bladder epithelia.

The high specific radioactivity that may be attained with ¹²⁵I or ¹³¹Ioffers tremendous therapeutic advantage if the radiation can be focusedon a localized target. Standard doses of oral or intravenous[¹³¹I]-sodium iodide for the treatment of thyroid malignancy can rangefrom 0.75 to 100 milliCi. As noted earlier, [125I] or[¹³¹I]-alkylcarboxamides may easily be synthesized at a specificactivity of 250 Ci/mmol or higher to give a compound with a specificactivity of greater than 1 Ci/mg. Injection or oral intake of 0.1 mg ofsuch compounds will yield therapeutic dose of ≧100 milliCi. Because thisradiation is selectively localized to hyperplastic or malignant cells,normal cells are spared and, I believe desirable therapeutic effects maybe achieved.

To carry out such therapeutic applications, the following procedures arecontemplated. The anti-tumor activity of a given [¹²⁵I] or[¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamide agonist/antagonist of theTRP-M8 receptor is first measured by its cell-inhibiting oranti-proliferative actions (versus the non-radioactive isotope) on celllines expressing the TRP-M8 receptor. If activity is found with EC50ranges of between nanomolar to low micromolar concentrations, then theradioactive compound will be tested in mice bearing transplanted tumorcell lines expressing the TRP-M8 receptor. Tumor volume, rate of growth,distant metastases, and histological features of the cancer cells innude mice will be evaluated using standard techniques that are wellknown in the art. Pre-clinical in vivo test results from the nude mousemodel and other animal models of cancer are the final prelude toclinical evaluation of the drug candidate in humans.

Before a [¹²³I] or [¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamideagonist/antagonist of TRP-M8 is administered to human cancer patients,the level of TRP-M8 expression in the target tissues preferably isdetermined. A standard polymerase-chain reaction of the mRNA for thereceptor may be used on biopsied tissues. Alternatively,[¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide radioreceptor binding to thebiopsied tissues may be measured.

The N-radioiodo-aryl-alkyl-alkylcarboxamide if administeredintravenously is subject to rapid degradation by liver amidases. One wayto circumvent this rapid biostransformation is to administer analternative amide substrate concurrently or just before the radioactivecarboxamide. Such substrates may be lidocaine (Xylocaine®) which can beinfused at a bolus dose of 50-100 mg over 2 to 3 min and this procedurerepeated twice for a total dose of 300 mg in one hr. Another drug inthis category is procainamide (Procan®). Procainamide can be given up to1.5 gm in a 6 hr period. These anti-arrhythmic cardiac drugs areconsidered relatively non-toxic at these doses and may be ideal forco-administration with radiodiagnostic procedures using [¹⁸F] or[¹²³I]-labeled TRP-M8 drugs or for radiotherapeutic doses of [¹²⁵I] or[¹³¹I]-labeled TRP-M8 drugs.

Another consideration in administering radiotherapeutic drugs to humancancer patients is that of toxicity. To avoid irradiation of the TRP-M8receptor in normal tissues, the drug can be delivered locally into thetumor (e.g. directly into the bladder) or into the regional circulationof the malignant tissues. If the radioactive drug is to be administeredby oral intake or by intravenous injection, it may be possible toprotect the TRP-M8 in normal tissues from the radiation by topical ororal administration of the non-radioactive drug. For example, thenon-radioactive ligand or a surrogate such as menthol may beadministered as a lozenge, in chewing gum, or as a capsule or pill, toprotect the mucous lining of the gastrointestinal tract against theradionuclide. Eye-drops and nose-drops containing the non-radioactiveligand may also be administered to protect the TRP-M8 receptors in thesetissues. In addition, radioprotective drugs such as thiols may beco-administered if the TRP-M8 receptor is present in tissues such as theliver or kidney.

Tests for Bioactivitiy and Anti-Neoplastic Actions on Bladder Cells. Thecompounds of this invention bind with high affinity (μg/mL or nanoM) tothe TRP-M8 receptor on biological membranes. Such affinities can bemeasured using radioreceptor assays and are familiar to pharmacologistsskilled in the art. Alternatively, certain cancer cell lines, such ashuman urothelial UROtsa cells (Master J. W. et al. Tissue culture modelof transitional cell carcinoma: characterization of twenty-twourothelial cell lines. Cancer Res 1986: 46:3630-3636, and Rossi, MR etal. The immortalized UROtsa cell line as a potential cell culture modelof human urothelium. Environ Health Perspect. 2001: 109:801-808), andprostate lymph node LNCaP cells, constitutively express functionalTRP-M8 binding sites on their membrane surfaces. The EC50 of candidatecompounds on calcum fluxes, cell growth, and proliferative activity insuch cells may be measured and Kd estimated. Potency andpharmacokinetics may then be optimized for lead candidates to take toanimal models of bladder cancer.

The conventional methods for evaluating the effect of a therapeuticagent for bladder cancer in animal models have been time and laborconsuming. First, the rodents have to be sacrificed, the bladders needto be inspected under the dissecting microscope, and a large number ofbladder tissue sections must be made throughout the entire bladder tohistologically evaluate the amount of tumor present. Moreover, only onetime point can be studied per rodent. Finally, to achieve anystatistically significant results, a large number of animals are neededand one could always question whether or not the tumor burden wassimilar in treated and untreated animals.

Zhou et al. (Visualizing superficial human bladder cancer cell growth invivo by green fluorescent protein expression Cancer Gene Therapy 2002:9, 681-686) have developed an elegant bioassay. Human bladder tumorcells (KU-7 cell line) stably expressing high levels of greenfluorescent protein (GFP) are transplanted into athymic mice byintravesical instillation. After about 1 week a small incision is madeto expose the bladder and its green fluorescence pixel area, whichrepresents the tumor burden, is quantified relative to the total area ofthe bladder. The incision is closed, but may be re-opened in each mouseand tumor progression over time can be quantified. It is possible tofollow in a given mouse the effectiveness of therapy. This is one methodfor assay of the contemplated invention. The candidate compound can betested in animals by oral administration, by parenteral injection(subeutaenous or intraperitoneal), or by intravesical instillation. Theamount of green fluorescence then predicts the amount of tumor growth inthe bladder.

Practice of the Invention. Preferred candidate compounds, whenadministered to humans, for example, by intravenous injection, arecleared rapidly from the bloodstream and concentrate in the urine. Sucha compound will then bind selectively to the TRP-M8 receptor on bladdercancer cells, deliver a lethal dose radiation to the cancer, and then beexcreted via the urine. For certain non-cancerous bladder conditions,such as interstitial cystitis, this destruction of the urothelium mayalso be desirable. To achieve optimal dose and safety, variousparameters affecting urine concentration of the candidate drug may beselected. For example, fluid intake can be regulated so that a knownvolume of urine is present in the bladder. After dosing, the drug may beflushed out by increased fluid intake or by using a diuretic such asfurosemide. An inhibitor of renal tubule re-uptake of the drug, such assulfinpyrazone, may be used to ensure that the radioactive drug is notre-absorbed once it is excreted into the urine. The presence of asoluble radiotherapeutic ensures that the bladder wall is uniformlyexposed to the drug (to help destroy microstatses) and avoids the needfor transurethral catheterization. It is well understood in the art ofcancer chemotherapy that a single agent may not be sufficient to controlthe growth and spread of disease. Thus, other agents may be used incombination with the present invention. Also, the precise dosage andduration of treatment is a function of the tissue being treated and canbe determined empirically using known testing protocols or byextrapolation from in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed formulations.

EXPERIMENTAL Example 1

Synthesis of N-haloarylcycloalkyl-alkylcarboxamide Agonists for TRP-M8Receptors (CPS-195) and Synthesis of Exemplary RadioactiveN-halo-aryl-alkyl-alkylcarboxamides

The acid chloride derivative of menthol required for preparation of theamides is available from optically pure menthol. Recent reports havesuggested that bi- and tricyclic amide derivatives of commerciallyavailable α-aminoalcohols may have optimal hydrophobicity and activity,and these amines will be coupled to the acid chloride. Halogen exchangeassisted by tetrakis(triphenylphosphine)palladium (O) will afford theiodinated aryl amide. The iodinated compound will be converted to thekey trimethylstannane intermediate, again on treatment with palladium(O) catalyst. The purified stannylated amide will be used to prepareboth ¹²⁵I-labelled and ¹⁸F-labelled reagents for radioreceptor assaysand PET imaging, respectively. Generation of electrophilic iodine bytreatment of radiolabelled sodium iodide with chloramines-T will allowpreparation of the ¹²⁵I-labelled material required for binding assays.Radioactive fluorine gas will be used to oxidize the carbon-tin bond togive the fluoroaromatic compound to be used in the PET imagingexperiments. Once we have information about first-generation ligandsfrom radioreceptor assays (vide infra), their structures will besystematically-modified to enhance binding, by changing substituents atthe 4′-position of the aryl ring.

Synthesis of 2-Isopropyl-5-methyl-cyclohexanecaboxylic acid[2-hydroxy-2-(3-hydroxy-phenyl)-ethyl]-methyl-amide. Phenyephrine HCl[(R)-(−)-3-(1-Hydroxy-2-methylamino-ethyl)-phenol. hydrochloride] waspurchased from Aldrich Chemicals, Co., Milwaukee, Wis. 1.0 g wasdissolved in 28 ml diethyether and 1 ml double-distilled water andcooled to 0° C. A pinch of the catalyst diaminopyrimidine was added.1.90 ml of p-menthoyl chloride was then added dropwise, followed by 2 mlof triethylamine. White precipitates appeared in the mixture, which wasstirred overnight at room temperature. The precipitate was dissolvedwith ethylacetate, washed with double-distilled water and dried oversodium sulfate. The organic phase was then evaporated under reducedpressure to yield the final product (1.8 g), which crystallized at roomtemperature. The expected molecular mass was then confirmed by massspectroscopy and the absorption spectrum by nuclear magnetic resonance.This compound was given the code of CPS-140.

[¹⁸F], [¹²³I], [¹²⁵I], and [¹³¹I]-N-halo-aryl-alkyl-alkylcarboxamideradioligands of the invention were synthesized, for example, at 25Ci/mmole. The non-radioactive forms of these chemicals are known to bepotent and active on the TRP-M8 receptor. For example, the fluorinatedanalog is active at nanomolar (10⁻⁹) in promoting calcium entry intoTRP-M8 transfected cells and into LNCaP (lymph node prostate cancercells) constitutively expressing these receptors.

A particularly preferred TRP-M8 receptor ligand embodiment of thepresent invention, sometimes designated CP-129, is illustrated byStructure 1 where the radioisotope is ¹²⁵I. The precursor of thiscompound isN-(2′-iodo-4′methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide.One can readily replace the non-radioactive iodine atom with one of theisotopes ¹⁸F, ¹²³I, ¹²⁵I, or ¹³¹I, the choice of which depends on theintended use.

CP-129 or its ¹²⁵I, ¹³¹I analogs are prepared from the nonradiolabeledtrimethyl tin precursor by oxidation with labeled sodium iodide andchloramine-T. The precursor is made from the parent by replacing theiodo group with a trimethyl tin group in the presence of tetrakis(triphenyl phosphine) palladium and bis(trimethyl)tin. The initialnonradiolabeled compound is prepared by reacting2-isopropyl-5-methylcyclohexane carbonyl chloride with2-iodo4-methoxylphenylamine (2-iodo-p-anisidine). It should be noted thecorresponding ¹⁸F compound may be made by the same technique.

A mixture ofN-(2′-iodo-4′methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide(500 mg, 1.25 mmol), tetrakis(triphenylphosphine)-palladium (150 mg,0.13 mmol, 10% molar equivalent), bis(trimethylstannyl) (510 mg, 1.5mmol), triethylamine (50 ml), and THF (50 ml) was heated at reflux for12 hr. The reaction mixture was evaporated to dryness in vacuum. Theresidue was dissolved in ethylacetate and crystallized by the additionof methanol.

Radiochemical synthesis to produce CP-129 used the following method. ATHF solution ofN-(2′-trimethylstannyl-4′-methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide(1 mg/ml) was prepared. To 5 ml of this solution was added Na¹²⁵I (0.5to 1.0 mCi, 3 to 5 ml) in 0.1 N NaOH, followed by the addition of 0.05 NHCl (10 ml) to adjust to pH 4.0 to 5.5. A freshly prepared solution (1ml) of chloramine-T (1 mg/ml) was added to the above mixture, and thesolution was incubated at room temperature for 15 min. After this time,20 ml of sodium metabisulfite (3 mg/ml) were added to terminate thereaction, and the solution was incubated for an additional 5 min.Finally, a saturated solution of sodium bicarbonate (50 ml) was added tothe reaction vial, and the radioactivity was extracted with chloroform(5 ml). The final product is obtained by HPLC chromatography and usedwithout carrier. For intravenous injection, the trimethyl tin compoundis supplied as a sterile ethanolic solution for reaction withradiolabeled NaI and chloramine-T in sterile saline. Unreacted materialsare removed simply using a C₁₈ Sep Pak cartridge, yielding CP-129 ofmore than 98 percent radiochemical purity. This is illustrated bySchematic 3.

The incorporation efficiency of radioactivity is nearly quantitative. Itshould be noted that, in the above synthesis, alkyl or cycloalkylsubstituents can be singly added onto the -aryl-alkyl ring using methodswell known to the art. The use of trimethyl tin reagents forradio-labeling is only one example of such technology for singleaddition of halogens, and alternative organometallic reagents areavailable.

Example 2

Radioreceptor Assay

The synthesized radioligand of the invention, such as the [¹²⁵I]-CP-129prepared as in Example 1, at a specific activity of 25 Ci/mmole, is nowused for a radioreceptor assay. In a standard test-tube method forcompetitive receptor binding, a tissue known to contain the TRP-M8receptor, such as dorsal root ganglia neuronal cultures or a humanprostate cancer cell line, is incubated with [¹²⁵I]-CP-129 untilsteady-state conditions are reached (usually 30 to 60 minutes). Thebound radioactive ligand is then separated from the free radioactiveligand by methods well known in the art such as filtration,centrifugation, dialysis, or size exclusion chromatography. Todifferentiate between specific (receptor) binding from non-specificbinding, a non-radioactive N-halo-aryl-alkyl-alkylcarboxamide, such asN-(3′-fluoro,4′-methoxyphenyl)-2-isopropyl-5-methyl-cyclohexanecarboxamide, may beused. After these parameters are established, the next procedure is toconduct a saturation experiment that will establish the Kd and the Bmax(which is the density of receptors in a given tissue and is apharmacological technique well known in the art). Various concentrationsof radioactive ligand are incubated with the receptor preparation andthe ratio of the bound and free levels of radioactive ligand ismeasured. The standard Rosenthal plot or Scatchard analysis of thebinding data yields the constants Kd and Bmax.

These and other uses of the present invention will become readilyapparent to the skilled artisan once he or she has read the disclosurein this application. It is to be understood that while the invention hasbeen described above in conjunction with preferred specific embodiments,the description and examples are intended to illustrate and not limitthe scope of the invention, which is defined by the scope of theappended claims.

1. A N-radioisotope-labeled-aryl-alkyl-alkylcarboxamide ligand whereinthe alkyl moiety of the alkylcarboxamide is a cycloalkane radical havingfrom 7 to about 14 carbons and with one to three C₁ to C₅ normal orbranched alkyl substituents, the radioligand having a high affinity toTRP-M8 receptors in cells and tissues and having a specific activity ofat least 20 Ci/mmol or greater, wherein the TRP-M8 affinity ischaracterized by a Kd of about 1×10⁻⁵ or less.
 2. The radioligand as inclaim 1 wherein the radioisotope label is covalently bound in themolecule.
 3. The radioligand as in claim 2 wherein the radioisotopelabel is selected from astatine, bromine, fluorine, iodine, astatide,bromide, fluoride, or iodide nadionuclides.
 4. The radioligand as inclaim 1 wherein the specific activity is about 20 Ci/mmol or greater andthe radioisotope moiety emits alpha, beta or gamma radiation.
 5. Acomposition comprising a radioligand, the radioligand being aN-radioistope-labeled-aryl-alkyl-alkylcarboxamide of Formula 1:R—(C═O)—N(H or CH₃)—R′—Y  Formula 1 where (a) R is a saturated ormonoethylenically unsaturated alkyl-substituted cyclic alkyl radicalcontaining a total of 7 to about 14 carbon atoms and is selected fromthe group consisting of cyclopentanes, cyclohexanes, cycloheptanes, andcyclooctanes, each cyclic alkyl radical containing from 1 to 3 C₁-C₅normal or branched alkyl substituents, (b) R′ is a normal or branchedC₁-C₃ carbon bridge with an optional hydroxy, and (c) Y is an aromaticradical containing one to four substituents of R₁ or R₂, and one tothree substituents of X, wherein R₁ is selected from the group hydrogen,hydroxyl, C₁-C₅ alkyl, C₁-C₃ alkoxy, C₁-C₃ carboxyalkyl, C₁-C₄carbonylalkylester, C₁-C₃ oxycarbonylalkyl, C₁-C₃ hydroxyalkyl, R₂ isselected from the group —SO₂NH-pyrimidine, —SO₃—(H, Me or Et), or—CH₂—SO₃—(H, Me or Et), acetyl, C₁-C₃ hydroxyalkyl, trifluoromethyl,nitro, cyano, halo, and X is selected from the group [¹⁸F]—, [¹²³I]—,[¹²⁵I]—, [¹³¹I]— [⁷⁶Br]— [⁷⁷Br]— and [²¹¹At]—.
 6. The composition as inclaim 5 wherein the cycloalkyl radical of the radioligand is((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl)-.
 7. The composition as inclaim 5 wherein the radioligand is a single enantiomer with its chiralcenter in R′.
 8. The composition as in claim 5 wherein the radioligandhas a specific activity of about 20 Ci/mmol or greater and emits alpha,beta or gamma radiation.
 9. The composition as in claim 5 wherein theradioligand is a ligand for the TRP-M8 receptor.
 10. A diagnosticmethod, comprising: providing a radioligand, the radioligand being aN-radioistope-labeled-aryl-alkyl-alkylcarboxamide of Formula 1:R—(C═O)—N(H or CH₃)—R′—Y  Formula 1 where (a) R is a saturated ormonoethylenically unsaturated alkyl-substituted cyclic alkyl radicalcontaining a total of 7 to about 14 carbon atoms and is selected fromthe group consisting of cyclopentanes, cyclohexanes, cycloheptanes, andcyclooctanes, each cyclic alkyl radical containing from 1 to 3 C₁-C₅normal or branched alkyl substituents, (b) R′ is a normal or branchedC₁-C₃ carbon bridge with an optional hydroxy, and (c) Y is an aromaticradical containing one to four substituents of R₁ or R₂, and one tothree substituents of a radio label X, wherein R₁ is selected from thegroup hydrogen, hydroxyl, C₁-C₅ alkyl, C₁-C₃ alkoxy, C₁-C₃ carboxyalkyl,C₁-C₄ carbonylalkylester, C₁-C₃ oxycarbonylalkyl, C₁-C₃ hydroxyalkyl, R₂is selected from the group —SO₂NH-pyrimidine, —SO₃—(H, Me or Et), or—CH₂—SO₃—(H, Me or Et), acetyl, C₁-C₃ hydroxyalkyl, trifluoromethyl,nitro, cyano, halo, and X is selected from the group [¹⁸F]—, [¹²³I]—,[¹²⁵I]—, [¹³¹I]— [⁷⁶Br]— [⁷⁷Br]— and [²¹¹At]—; contacting theradioligand with cells or tissues under conditions sufficient to permitspecific binding between the radioligand and TRP-M8 receptors if saidreceptors are carried by the cells or tissues; and, examining the cellsor tissues for presence of the radio lable X.